Sjogren's syndrome (SS) is a chronic autoimmune disease which results in decreased secretion from salivary and lacrimal glands. Although intense lymphocytic infiltration and epithelial cell destruction are characteristic of the disease, identifying the actual mechanism(s) of salivary epithelial injury in SS remains a major challenge. The broad, long-term objectives of this proposal are to define salivary gland epithelial cell apoptosis in SS, and to address the role of granule-mediated cellular cytotoxicity in this process. The specific aims of the proposal are: (1) to quantify and characterize salivary gland epithelial cell apoptosis in SS, and to demonstrate the novel autoantigen fragments specifically generated by the granule-mediated cytotoxicity pathway in SS salivary glands; (2) to use the human salivary gland cell line (HSG) as a model system to study the consequences of cytolytic granule-induced apoptosis on autoantigen structure and distribution; (3) to identify the sites at which granzyme B cleaves La; and (4) to address immunogenicity of novel La fragments in vitro. Apoptosis in salivary gland biopsies from SS patients will be quantitated using the TUNEL assay, and will be correlated with the lymphocytic infiltrate, both in terms of distribution and magnitude. Vectorial distribution of cytolytic T cell granules will be sought in SS salivary glands. The apoptotic pathways induced by ultraviolet irradiation and cytolytic granule contents in HSG cells and HeLa cells will be compared. The granzyme B cleavage site in La will be defined by radiosequencing or mutational analysis. Recombinant proteins corresponding to the intact protein and the granzyme B cleavage fragments will be generated, and their immunogenicity will be addressed using an in vitro immunogenicity assay. These studies will yield an enhanced understanding of the relevance of granule-mediated apoptosis in the pathogenesis of SS and other autoimmune diseases.